Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose

Pseudomonas cichoriii D-tagatose 3-epimerase (P. cichorii D-TE) can efficiently catalyze the epimerization of not only D-tagatose to D-sorbose, but also D-fructose to D-psicose, and is used for the production Of D-psicose from D-fructose. The crystal structures of R cichorii D-TE alone and in complexes with D-tagatose and D-fructose were determined at resolutions of 1.79, 2.28, and 2.06 angstrom, respectively. A subunit of P. cichorii D-TE adopts a (beta/alpha)(8) barrel structure, and a metal ion (Mn2+) found in the active site is coordinated by Glu152, Asp185, His211, and Glu246 at the end of the beta-barrel. P. cichorii D-TE forms a stable dimer to give a favorable accessible surface for substrate binding on the front side of the dimer. The simulated omit map indicates that 02 and 03 Of D-tagatose and/or D-fructose coordinate Mn2+ and that C3-O3 is located between carboxyl groups of Glul52 and Glu246, supporting the previously proposed mechanism of deprotonation/protonation at C3 by two Glu residues. Although the electron density is poor at the 4-, 5-, and 6-positions of the substrates, substrate-enzyme interactions can be deduced from the significant electron density at O6. The O6 possibly interacts with Cys66 via hydrogen bonding, whereas 04 and 05 in D-tagatose and 04 in D-fructose do not undergo hydrogen bonding to the enzyme and are in a hydrophobic environment created by Phe7, Trp15, Trp113, and Phe248. Due to the lack of specific interactions between the enzyme and its substrates at the 4- and 5-positions, P. cichorii D-TE loosely recognizes substrates in this region, allowing it to efficiently catalyze the epimerization Of D-tagatose and D-fructose (C4 epimer Of D-tagatose) as well. Furthermore, a C3-O3 proton-exchange mechanism for P. cichorii D-TE is suggested by X-ray structural analysis, providing a clear explanation for the regulation of the ionization state of Glu152 and Glu246. (c) 2007 Elsevier Ltd. All rights reserved.